Wild type enrichment of a 25-phospho-steroid kinase involved in anaerobic steroid degradation

The anaerobic degradation of cholesterol fundamentally differs from the cytochrome P450 monooxygenase dependent activation of the isoprenoid side chain at C26 in aerobes. In the denitrifying β-proteobacterium Sterolibacterium denitrificans, activation of primary C26 proceeds via a three-step cascade involving (i) a water-dependent hydroxylation at tertiary C25, (ii) an ATP-dependent dehydration to an alkene, and (iii) a water-dependent hydroxylation at primary C26 to an allylic alcohol. Here, we enriched a 25-hydroxy-steroid kinase (25-HSK) from Sterolibacterium denitrificans that catalyses the ATP-dependent transformation of 25-OH-cholest-1,4-diene-3-one (25-OH-CDO) to 25-phospho-CDO.